Methods for determining total body skeletal muscle mass

ABSTRACT

The present invention is based on the finding that enrichment of D3-creatinine in a urine sample following oral administration of a single defined dose of D3-creatine can be used to calculate total-body creatine pool size and total body skeletal muscle mass in a subject. The invention further encompasses methods for detecting creatinine and D3-creatinine in a single sample. The methods of the invention find use, inter alia, in diagnosing disorders related to skeletal muscle mass, and in screening potential therapeutic agents to determine their effects on muscle mass.

FIELD OF THE INVENTION

This invention relates to methods for determining the total body pool size of creatine and total body skeletal muscle mass in a subject by the use of an orally administered tracer dose of D3-creatine, and encompasses improved methods for determining the concentration of creatinine in a biological sample.

BACKGROUND OF THE INVENTION

Skeletal muscle plays a central role in metabolic adaptations to increasing and decreasing physical activity, in disease (e.g. cachexia), in obesity, and in aging (e.g. sarcopenia). Sarcopenia is described as the age-associated loss of skeletal muscle (Evans (1995) J. Gerontol. 50A:5-8) and has been associated with mobility disability (Janssen and Ross, (2005) J. Nutr. Health Aging 9:408-19) and greatly increased health-care costs for elderly people (Janssen et al. (2004) J. Am. Geriatr. Soc. 52:80-5). Loss of skeletal muscle with advancing age is associated with decreased energy requirements and concomitant increase in body fatness, weakness and disability, insulin resistance and risk of diabetes. Loss of skeletal muscle associated with an underlying illness (cachexia) is associated with a greatly increased mortality (Evans (2008) Clin. Nutr. 27:793-9).

Because of the important role total body skeletal muscle mass plays in aging and disease, there is an effort in the pharmaceutical arts to identify therapeutic agents that will stimulate muscle protein synthesis and increase muscle mass. However, current methodologies for quantification of muscle synthesis and muscle mass often involve invasive procedures (e.g. muscle biopsies) or rely on expensive equipment (i.e. DEXA, MRI, or CT) that provides only indirect data on whole body muscle mass. Because of these limitations, no method is routinely used in the clinic for estimation of skeletal muscle mass, and no diagnostic criteria for estimates of muscle mass have been produced. As a result, there is a no straightforward way to determine the effects of potential therapeutic agents on muscle protein synthesis mass.

Accordingly, there remains a need in the art for reliable, easily-performed, non-invasive measurements of total body skeletal muscle mass.

BRIEF SUMMARY OF INVENTION

The present invention is based on the finding that steady-state enrichment of D3-creatinine in a urine sample following oral administration of a single defined tracer dose of D3-creatine can be used to calculate total-body creatine pool size and skeletal muscle mass in a subject.

The invention is further based on the finding that the concentration of creatinine in a biological sample can be determined by measuring the concentration of creatinine M+2 isotope and dividing this concentration by a dilution factor, where the dilution factor is the ratio of the concentration of creatinine M+2 to the concentration of creatinine M+0 in the biological sample. Determining the creatinine concentration in a biological sample according to these improved methods allows for the simultaneous measurement of the concentration of creatinine and D3-creatinine in a single sample using widely-available instrumentation. Accordingly, this improved detection method will facilitate the wide-spread adaptation of the present methods for use in determining skeletal muscle mass in patients.

Accordingly, in one aspect the invention provides a method for determining the total body skeletal muscle mass in a subject, where the method comprises the steps of:

-   -   (a) orally administering 10-200 mg D3-creatine or a salt or         hydrate thereof to the subject;     -   (b) allowing at least 12 hours to elapse after the         administration of the D3-creatine;     -   (c) obtaining a biological sample from the subject,     -   (d) determining the concentration of creatinine and         D3-creatinine in said biological sample;     -   (e) using the creatinine and D3-creatinine concentrations         determined in step     -   (f) to calculate the total body skeletal muscle mass of the         subject.

In particular embodiments, the biological sample is a urine sample.

In certain embodiments, the concentration of creatinine and D3-creatinine in the urine sample is determined by HPLC/MS/MS.

In another aspect, the invention provides a method of determining the concentration of creatinine in a biological sample from a subject, said method comprising the steps of:

-   -   (a) obtaining a biological sample from the subject;     -   (b) analyzing the biological sample to determine the peak area         of the creatinine M+2 isotope peak for the biological sample;     -   (c) comparing the peak area determined in step (b) to a         calibration curve generated using D3-creatinine to determine the         concentration of the creatinine M+2 isotope in the biological         sample;     -   (d) dividing the concentration obtained in step (c) by a         dilution factor, where the dilution factor is the ratio of the         concentration of creatinine M+2 to the concentration of         creatinine M+0 in the biological sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Urinary D3-creatinine enrichment and total body creatine pool size in growing rats. (A) Urinary D3-creatinine enrichment (determined by isotope ratio mass spectrometry) in 9 week-old (mean body weight 304±11 g, n=10) and 17 week-old (mean body weight 553±39 g, n=10) rats at the indicated time after a single oral 0.475 mg dose of D3-creatine, showing achievement of isotopic steady state by 48 h, and clear separation of growing rat age groups (P<0.001 between groups at all times; within groups, the difference between 48 and 72 h is not significant; 2-factor ANOVA and Student's t test). (B) Creatine pool size calculated from 72 h urinary D3-creatinine enrichments for the rat groups in FIG. 1, showing clear separation of age groups (p<0.0001).

FIG. 2. Correlation between Lean Body Mass by Quantitative Magnetic Resonance and total body creatine pool size, adjusted for age effect, for the rat groups in FIG. 1 (r_(all rats)=0.69; P<0.001).

FIG. 3. Even within the rat groups of different age from FIG. 1, there is a significant correlation of creatine pool size and lean body mass by either quantitative magnetic resonance (left) or DEXA (right).

FIG. 4. Significant correlation between lean body mass determined by quantitative magnetic resonance and creatine pool size determined by D3-creatine dilution in 22 week-old rats (n=10 per group) treated the previous two weeks with either vehicle or dexamethasone (P<0.001 and P=0.01, respectively).

FIG. 5. Correlation between lean body mass determined by quantitative magnetic resonance and total-body creatine pool size determined by D3-creatine dilution for all 40 rats used in the two cross-sectional studies (y=0.20x+91.6; r=0.9517; P<0.0001).

FIG. 6. This figure shows a flow chart for one embodiment of the method of determining total body skeletal mass.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding that enrichment of D3-creatinine in a urine sample following oral administration of a single defined dose of D3-creatine can be used to calculate total-body creatine pool size and skeletal muscle mass in a subject. Accordingly, the invention provides a non-invasive, accurate method of determining total body skeletal muscle. The methods of the invention find use, inter alia, in diagnosing and monitoring medical conditions associated with changes in total body skeletal muscle mass, and in screening potential therapeutic agents to determine their effects on muscle mass.

According to the method, D3-creatine is orally administered to a subject. Although the present is not limited by mechanism, it is believed that the D3-creatine is rapidly absorbed, distributed, and actively transported into skeletal muscle, where it is diluted in the skeletal muscle pool of creatine. Skeletal muscle contains the vast majority (>than 98%) of total-body creatine. In muscle tissue, creatine is converted to creatinine by an irreversible, non-enzymatic reaction at a stable rate of about 1.7% per day. This creatinine is a stable metabolite that rapidly diffuses from muscle, is not a substrate for the creatine transporter and cannot be transported back into muscle, and is excreted in urine. As a result, once an isotopic steady-state is reached, the enrichment of a D3-creatinine in spot urine sample after a defined oral tracer dose of a D3 creatine reflects muscle creatine enrichment and can be used to directly determine creatine pool size. Skeletal muscle mass can then be calculated based on known muscle creatine content.

Accordingly, in one aspect the invention provides a method of determining the total body skeletal muscle mass in a subject, where the method comprises the steps of:

-   -   (a) orally administering 10-200 mg D3-creatine or a salt or         hydrate thereof to the subject;     -   (b) allowing at least 12 hours to elapse after the         administration of the D3-creatine;     -   (c) obtaining a urine sample from the subject,     -   (d) determining the concentration of creatinine and         D3-creatinine in said urine sample;     -   (e) using the creatinine and D3-creatinine concentrations         determined in step     -   (f) to calculate the total body skeletal muscle mass of the         subject.

In certain embodiments, a hydrate of D3-creatine is administered to the subject. In particular embodiments, D3-creatine monohydrate is administered.

The dose of D3-creatine to be administered to the subject is preferably selected such that the labeled creatine is rapidly absorbed into the bloodstream and spillage of excess label into the urine is minimized. Accordingly, for a human subject the dose of D3-creatine is typically 5-250 mgs, such as 20-125 mgs. In particular embodiments, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mgs of D3-creatine is administered. In some embodiments, the dose is adjusted based on the gender of the subject. Thus, in certain embodiments, the subject is female and 10-50, such as 20-40, or more particularly, 30 mg of D3-creatine is administered to the subject. In other embodiments, the subject is male and 40-80 mg, such as 50-70, or more particularly, 60 mg or 70 mg of D3-creatine is administered to the subject.

Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions, each with aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions. For instance, for oral administration in the form of a tablet or capsule, the active drug component may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Generally, powders are prepared by comminuting the compound to a suitable fine size and mixing with an appropriate pharmaceutical carrier such as an edible carbohydrate, as, for example, starch or mannitol. Flavorings, preservatives, dispersing agents, and coloring agents may also be present.

Capsules can be made by preparing a powder, liquid, or suspension mixture and encapsulating with gelatin or some other appropriate shell material. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol may be added to the mixture before the encapsulation. A disintegrating or solubilizing agent such as agar-agar, calcium carbonate or sodium carbonate may also be added to improve the availability of the medicament when the capsule is ingested. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents may also be incorporated into the mixture. Examples of suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants useful in these dosage forms include, for example, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

Tablets can be formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant, and pressing into tablets. A powder mixture may be prepared by mixing the compound, suitably comminuted, with a diluent or base as described above. Optional ingredients include binders such as carboxymethylcellulose, aliginates, gelatins, or polyvinyl pyrrolidone, solution retardants such as paraffin, resorption accelerators such as a quaternary salt, and/or absorption agents such as bentonite, kaolin, or dicalcium phosphate. The powder mixture may be wet-granulated with a binder such as syrup, starch paste, acadia mucilage or solutions of cellulosic or polymeric materials, and forcing through a screen. As an alternative to granulating, the powder mixture may be run through the tablet machine and the result is imperfectly formed slugs broken into granules. The granules may be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc or mineral oil. The lubricated mixture is then compressed into tablets. The compounds of the present invention may also be combined with a free flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material, and a polish coating of wax may be provided. Dyestuffs may be added to these coatings to distinguish different unit dosages.

Oral fluids such as solutions, syrups, and elixirs may be prepared in dosage unit form so that a given quantity contains a predetermined amount of the compound. Syrups may be prepared, for example, by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic alcoholic vehicle. Suspensions may be formulated generally by dispersing the compound in a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol ethers may be added. Solubilizers that may be used according to the present invention include Cremophor EL, vitamin E, PEG, and Solutol. Preservatives and/or flavor additives such as peppermint oil, or natural sweeteners, saccharin, or other artificial sweeteners; and the like may also be added.

According to the method, the urine sample in preferably collected after enrichment levels of D3-creatinine in the urine have reached a steady-state. Thus in one embodiment, at least 6 hours or at least 12 hours is allowed to elapse after the administration of the D3-creatine but prior to the collection of the urine sample. In certain embodiments, at least 24 hours is allowed to elapse. In particular embodiments, at least 36 hours, at least 48 hours, at least 60 hours, or at least 72 hours are allowed to elapse after the administration of the D3-creatine and before the collection of the urine sample.

The invention also encompasses certain improved analytic methods for detecting creatinine and D3-creatinine in urine samples. Specifically, the invention provides for the detection of creatinine and D3-creatinine in urine samples by HPLC/MS, particularly HPLC/MS/MS. However, alternate methods know in the art may also be used to detect creatinine and/or D3 creatinine in urine samples. Such methods include direct or indirect colorimetric measurements, the Jaffe method, enzymatic degradation analysis, or derivatization of the creatinine followed by GC/MS analysis of HPLC with fluorescence detection.

Thus in one aspect, the invention provides a method of determining the concentration of creatinine in a biological sample from a subject, said method comprising the steps of:

-   -   (a) obtaining a biological sample from the subject;     -   (b) analyzing the biological sample to determine the peak area         of the creatinine M+2 isotope peak for the biological sample;     -   (c) comparing the peak area determined in step (b) to a         calibration curve generated using D3-creatinine to determine the         concentration of the creatinine M+2 isotope in the biological         sample;     -   (d) dividing the concentration obtained in step (c) by a         dilution factor, where the dilution factor is the ratio of the         concentration of creatinine M+2 to the concentration of         creatinine M+0 in the biological sample.

The biological sample may be any appropriate sample including, but not limited to, urine, blood, serum, plasma, or tissue. In one particular embodiment, the biological sample is a urine sample. In another particular embodiment, the biological sample is a blood sample.

In a preferred embodiment, the peak area of the creatinine M+2 isotope peak is determined using liquid chromatography/mass spectroscopy (LC/MS/MS).

In one embodiment, the dilution factor is 0.0002142±0.0000214. More particularly, the dilution factor is 0.0002142±0.00001, such as 0.0002142±0.000005.

The methods of the invention are useful for diagnosing and monitoring medical conditions associated with changes in total body skeletal muscle mass. Examples of medical conditions in which loss of muscle mass plays an important role in function, performance status, or survival include, but are not limited to frailty and sarcopenia in the elderly; cachexia (e.g., associated with cancer, chronic obstructive pulmonary disease (COPD), heart failure, HIV-infection, tuberculosis, end stage renal disease (ESRD); muscle wasting associated with HIV therapy, disorders involving mobility disability (e.g., arthritis, chronic lung disease); neuromuscular diseases (e.g., stroke, amyotrophic lateral sclerosis); rehabilitation after trauma, surgery (including hip-replacement surgery), medical illnesses or other conditions requiring bed-rest; recovery from catabolic illnesses such as infectious or neoplastic conditions; metabolic or hormonal disorders (e.g., diabetes mellitus, hypogonadal states, thyroid disease); response to medications (e.g., glucocorticoids, thyroid hormone); malnutrition or voluntary weight loss. The claimed methods are also useful in sports-related assessments of total body skeletal muscle mass.

The methods of the invention are also useful for screening test compounds to identify therapeutic compounds that increase total body skeletal muscle mass. According to this embodiment, the total body skeletal mass of a subject is measured according to the method before and after a test compound is administered to the subject. The assessment of total body skeletal muscle mass can be repeated at appropriate intervals to monitor the effect of the test compound on total body skeletal muscle mass.

EXPERIMENTAL

Use of the D3-creatine Tracer Dilution Method to Determine Total Body Skeletal Muscle Mass in a Pre-Clinical Model

A dose of 0.475 mg D3-creatine per rat was determined to be rapidly and completely absorbed and reach the systemic circulation with minimal urinary spillage, such that >99% of the D3-creatine tracer dose should be available to equilibrate with the body creatine pool.

The creatine dilution method was then used to determine urinary D3-creatine enrichment and the time to isotopic steady state in growing rats. In a cross-sectional study, a single oral dose of 0.475 mg D3-creatine per rat was given to two groups of rats, 9 and 17 weeks of age, and urine was collected at 24, 48, and 72 hour time points after dosing. As expected, the larger, older rats had lower urinary D3-creatinine enrichment (expressed as mole percent excess, MPE) at all time points than the younger, smaller rats, reflecting greater dilution of the D3-creatine tracer in the total body creatine pool. For both age groups, urinary enrichment was highest at 24 h and stable between 48 and 72 h, indicating isotopic steady state was achieved between 24 and 48 h after the tracer D3-creatine dose. (FIG. 1A).

Total body creatine pool size was then calculated using a formula for determination of pool size based on enrichment of a tracer, assuming a single creatine pool (Wolfe and Chinkes (2005) Calculation of substrate kinetics: Single-pool model. 2nd ed. Isotope tracers in metabolic research. Hoboken, N.J.: John Wiley & Sons, Inc. 21-9): the D3-creatine dose (0.475 mg) was divided by the D3-creatinine enrichment (MPE/100). FIG. 1B shows the total body creatine pool sizes calculated from urinary enrichment 72 h after the tracer dose for the 9 and 17 week-old rat groups and indicates the creatine pool size for the larger, older rats is significantly larger than for the smaller, younger rats.

The day before giving the tracer dose of D3-creatine, lean body mass (LBM) in all rats was assessed by either quantitative magnetic resonance (QMR) or DEXA. FIG. 2 shows that after accounting for age effect, LBM by QMR and creatine pool size are significantly correlated. LBM by QMR and creatine pool size are also significantly correlated within each age group, and LBM by DEXA and creatine pool size are significantly correlated within the 17 week-old age group (FIG. 3).

In a second cross-sectional study, an older rat age group (still within the rat growth phase of 22 weeks of age) was treated once daily subcutaneously with either saline vehicle, or dexamethasone to induce skeletal muscle atrophy for 2 weeks prior the administration of D3-creatine. As with the first cross-sectional study with 9 and 17 week-old rats, isotopic steady state was reached between 48 and 72 h.

Compared to vehicle-treated controls, dexamethasone induced a significant reduction in LBM (353±32 vs. 459±45 g, P<0.001) and a significant reduction in total body creatine pool size (1216±227 vs. 1853±228 mg, P<0.001). As in the first study, LBM and creatine pool size were significantly correlated within the two individual treatment groups (FIG. 4).

FIG. 5 show the correlation between LBM and creatine pool size for all 40 rats used in the two cross-sectional studies (r=0.95; P<0.001).

Use of the D3-creatine Tracer Dilution Method to Determine Total Body Skeletal Muscle Mass in Human Subjects

Human subjects are orally administered a single dose of 30, 60, or 100 mgs of D3 creatine-monohydrate. Urine samples are then collected 1, 2, 3, 4, 5,or 6 days after administration of the D3-creatine monohydrate dose.

Urine pharmacokinetic analyses for each collection interval may include quantitation of MPE ratio by IRMS, ratio of deuterium-labeled creatine+deuterium-labeled creatinine to total creatine+total creatinine by LCMS, total creatinine, creatine pool size, and % of deuterium-labeled creatine dose excreted in urine.

Steady-state enrichment (MPE) can be assessed both visually and from the estimate of the slope from the linear regression of enrichment (MPE) vs time (midpoint of each urine collection interval). A mixed effect ANOVA model can be fit with time (continuous variable) as a fixed effect and subject as a random effect. The coefficient for the slope of the time effect can be used to evaluate steady-state. The 90% confidence intervals for the slope can be calculated.

Creatine pool size can be estimated once steady-state enrichment has been achieved a for each collection interval during steady-state according to the formula: [Amount of D3 Cr dosed (g)−total Amount of urinary D3 Cr(0−t) (g)]/enrichment ratio(t) where t is the urine collection interval during steady-state.

Muscle mass can be estimated from the creatine pool size by assuming that the creatine concentration is 4.3 g/kg of whole wet muscle mass (WWM) (Kreisberg (1970) J Appl Physiol 28:264-7).

Muscle mass=creatine pool size/Cr concentration in muscle

Creatine pool size can also be estimated by total urine creatinine (moles/day) divided by K (1/day).

The excretion rate constant (K) can be estimated using a rate excretion method by estimating the declining slope of the line for the log of the amount of D3-creatine in urine collection interval vs. time (midpoint of that urine collection interval) for each collection interval over time. This estimate of K can be used in calculating creatine pool size from 24 hr urinary creatinine excretion rather than using an estimate of turnover form the literature.

Analytic Methods for Quantitating D3-creatine and D3-creatinine in Urine Samples from Clinical Subjects

Reference Standards of D3-Creatine monohydrate and D3-creatinine were purchased from C/D/N Isotopes, Montreal Canada.

HPLC-MS/MS Analysis

The separation of D3-creatine was carried out using an Acquity UPLC (Waters Corp., Milford, Ma.) equipped with a Zorbax Hilic Plus silica analytical column (50×2.1 mm, Rapid Resolution HD 1.8μ, Agilent Corp., Santa Clara Calif.). Injection volume is typically 8 μL.

Mobile phase A (MP A) consisted of 10 mM ammonium formate in water and mobile phase B is acetonitrile. Gradient chromatography was employed with initial mobile phase composition of 2% 10 mM ammonium formate with a flow rate of 0.7 mL/min. This was held for 0.5 minute and then a linear gradient to 50% MPA was achieved at 2.3 minutes. This was immediately increased to 80% and held for 0.4 minutes and then returned to starting conditions at 2.9 minutes. The total run time was 3.5 minutes. This gradient allowed baseline separation of the D3-creatine from interfering compounds.

The detection of D3-creatine was carried out using a Sciex API5000 (Applied Biosystems, Foster City, Calif.). The HPLC system was connected to the API5000 through a turbo ion spray source operating in positive ionization mode using the following parameters: ionization temperature of 650° C., ionspray voltage of 2500 V, curtain gas setting of 45 (N₂), nebulizer gas setting was 65 (N₂), drying gas setting was 70 (N₂), collision gas setting of 3 (N₂). All other mass spectrometer parameters were optimized for the individual transitions. The following ion transitions (MRM) were acquired: D3-creatine is m/z=135 to m/z=47 with a typical retention time of 1.99 min. The creatine standard is monitored with an ion transition of m/z=139 to m/z=50 with a typical retention time of 1.99 min.

The separation of the creatinine and D3-creatinine analytes were carried out using an Acquity UPLC (Waters Corp., Milford, Mass.) equipped with a Zorbax Hilic Plus silica analytical column , dimensions of 50×2.1 mm (Rapid Resolution HD 1.8μ, Agilent Corp., Santa Clara Calif.). Injection volume was typically 5 μL.

Mobile phase A consisted of 5 mM ammonium formate and mobile phase B was acetonitrile. Gradient chromatography was employed with initial mobile phase composition of 2% 5 mM ammonium formate with a flow rate of 0.7 mL/min. This was held for 0.4 minute and then a linear gradient to 40% MPA was achieved at 2.1 minutes. This was immediately increased to 50% at 2.2 minutes and held for 0.4 minutes and then returned to starting conditions at 2.7 minutes. The total run time was 3.5 minutes. This gradient allowed baseline separation of the d3-creatinine and creatinine from interfering compounds.

The detection of the creatinine and D3-creatinine analytes was carried out using a Sciex API5000 (Applied Biosystems, Foster City, Calif.). The HPLC system was connected to the API5000 through a turbo ion spray source operating in positive ionization mode using the following parameters: ionization temperature of 350° C., ionspray voltage of 5500 V, curtain gas setting of 45 (N₂), nebulizer gas setting was 60 (N₂), drying gas setting was 65 (N₂), collision gas setting of 3 (N₂). All other mass spectrometer parameters were optimized for the individual transitions. The following ion transitions (MRM) were acquired: D3-creatinine is m/z=117 to m/z=47 and for creatinine (M+2 isotope) was m/z=116 to m/z=44 with a typical retention time of 1.5 min. The creatine standard is monitored with an ion transition of m/z=121 to m/z=51 with a typical retention time of 1.5 min. For creatinine, the M+2 isotope version was acquired to avoid diluting the sample with buffer.

Endogenous creatinine concentration values are determined in human urine clinical samples using a D3-creatinine calibration standard curve. The D3-creatinine isotope behaves similarly to creatinine throughout the extraction and HPLC-MS/MS procedures, thus allowing clean urine matrix to prepare standards and QC samples.

The amount of endogenous creatinine (m/z=114) in the human clinical samples is much greater (1000 fold) than the levels of D3-creatinine. Therefore, instead of diluting the sample, the M+2 isotope of creatinine (m/z=116) will be monitored, thus allowing the simultaneous measurement of creatinine and D3-creatinine from one sample analysis.

The MRM of (M+2) endogenous creatinine (116/44) is monitored. A correction factor that represents the ratio of the MRM of 116/44 to 114/44, is used to correct the calculated concentrations determined from the d3-creatinine calibration curve. The isotope ratio (M+2) MRM/(M+0) MRM or correction factor is 0.00286. Therefore, the amount of D3-creatinine, which would come from the D3 creatine dose and the endogenous creatinine, can be quantitated from the single D3-creatinine calibration curve.

EXAMPLE

Chemical and Reagents: Acetonitrile and Water (all HPLC grade or better) purchased from Sigma Aldrich (St. Louis, Mo.). Ammonium Formate purchased from Sigma Aldrich (St. Louis, Mo.). Reference Standards of d3-Creatine (monohydrate) and d3-creatinine were purchased from CDN Isotopes, Montreal Canada.

Stock solutions of d3-creatine and d3-creatinine are prepared at 1.0 mg/mL in water and confirmation of equivalence is performed. Dilute solutions ranging from 0.1 μg/mL to 100 μg/mL and 0.2 μg/mL to 200 μg/mL are prepared in water and used to prepare calibration standards and quality control (QC) samples in human urine for d3-creatine and d3-creatinine, respectively. Isotopically labelled internal standards for creatine (SIL) (¹³C₃ ²H₃ ¹⁵N₁-creatine) and creatinine (SIL) (¹³C₃ ²H₄ ¹⁵N₁-creatinine) are prepared at 1.0 mg/mL in water. Dilute solutions of these are prepared at 500 ng/mL in acetonitrile and used as an extraction solvent for the urine standards, quality controls and study samples.

Sample Preparation: (d3-creatine, creatinine and d3-creatinine in urine) A 200 μL aliquot of the internal standard working solution (500 ng/mL) in acetonitrile is added to each well, except double blank samples, acetonitrile is added. A 40 μL aliquot of sample, standard or QC is transferred to the appropriate wells in the plate containing the SIL. The plate is sealed and vortex mixed for approximately 3 minutes. The plate is centrifuged at approximately 3000 g for 5 minutes. Supernatant is transferred to a clean 96 well plate and then injected onto the HPLC-MS/MS system for analysis. D3-creatine and d3-creatinine are analyzed from separate human urine samples.

HPLC-MS/MS Analysis

The separation of d3-creatine, d3-creatinine and creatinine is carried out using an Acquity UPLC (Waters Corp., Milford, Mass.) equipped with a Agilent Zorbax Hilic Plus silica analytical column, dimensions of 50×2.1 mm (Rapid Resolution HD 1.8μ, Agilent Corp., Santa Clara Calif.). Injection volume is typically 2 μL.

D3-creatine: mobile phase A consists of 10 mM ammonium formate and mobile phase B is acetonitrile. Gradient chromatography is employed with initial mobile phase composition at 2% 10 mM ammonium formate with a flow rate of 0.7 mL/min. This is held for 0.5 minute and then a linear gradient to 50% MPA is achieved at 2.3 minutes. This is increased to 80% over 0.2 minutes and held for 0.4 minutes and then returned to starting conditions at 3.0 minutes. The total run time is 3.5 minutes.

The detection of d3-creatine is carried out using a Sciex API5000 (Applied Biosystems, Foster City, Calif.). The HPLC system is connected to the API5000 through a turbo ion spray source operating in positive ionization mode using the following parameters: ionization temperature of 650° C., ionspray voltage of 2500 V, curtain gas setting of 45 (N₂), nebulizer gas setting is 65 (N₂), drying gas setting is 70 (N₂), collision gas setting of 3 (N₂). All other mass spectrometer parameters are optimized for the individual transitions. The following ion transitions (MRM) are acquired: d3-creatine is m/z=135 to m/z=47 with a typical retention time of 2 min. The SIL is monitored with an ion transition of m/z=139 to m/z=50 with a typical retention time of 2 min.

D3-creatinine: mobile phase A consisted of 5 mM ammonium formate, and mobile phase B is acetonitrile. Gradient chromatography is employed with initial mobile phase composition at 2% 5 mM ammonium formate with a flow rate of 0.7 mL/min. This is held for 0.4 minute and then a linear gradient to 60% acetonitrile is achieved at 2.1 minutes. This is immediately increased to 50% acetonitrile and held for 0.4 minutes and then returned to starting conditions at 2.7 minutes. The total run time is 3.5 minutes. The detection of the creatinine and d3-creatinine analytes is carried out using a Sciex API5000 (Applied Biosystems, Foster City, Calif.). The HPLC system was connected to the API5000 through a turbo ion spray source operating in positive ionization mode using the following parameters: ionization temperature of 350° C., ionspray voltage of 5500 V, curtain gas setting of 45 (N₂), nebulizer gas setting was 60 (N₂), drying gas setting was 65 (N₂), collision gas setting of 3 (N₂). All other mass spectrometer parameters are optimized for the individual transitions. The following ion transitions (MRM) are acquired: d3-creatinine is m/z=117 to m/z=47 and for creatinine (M+2 isotope) is m/z=116 to m/z=44 with a typical retention time of 1.5 min. The SIL is monitored with an ion transition of m/z=121 to m/z=51 with a typical retention time of 1.5 min. For creatinine, the M+2 isotope MRM is acquired to avoid diluting the sample with a surrogate matrix (a creatinine free control urine is not available). These isotopes will behave similarly throughout the extraction and HPLC-MS/MS procedures, thus allowing clean urine matrix to prepare standards and QC samples as well as allowing for the quantification of endogenous creatinine using a calibration curve that was generated from the deuterated form of creatinine. Therefore, the amount of d3-creatinine and the endogenous creatinine, can be quantitated from the single d3-creatinine calibration curve.

HPLC-MS/MS data were acquired and processed (integrated) using Analyst™ software (Version 1.4.2, MDS Sciex, Canada). A calibration plot of area ratio versus d3-creatinine concentration was constructed and a weighted 1/x² linear regression applied to the data.

Results

To perform bioanalytical quantification of biomarkers using LC/MS/MS, a surrogate matrix or a surrogate analyte must be used. In this assay, human urine can be used since d3-creatinine is not found endogenously and the quantification of creatinine can be determined from the d3-creatinine calibration curve. The equivalency of d3-creatinine and creatinine is shown.

D3-creatinine and Creatinine Equivalence Determination

A number of experiments were performed in order to verify that d3 creatinine can be used as a surrogate analyte to quantitate creatinine and that the MRM transition of 116/44 (M+2) can be used with the isotope ratio correction factor. To confirm that d3 creatinine can be used as a surrogate analyte for creatinine; two concentration levels of creatinine and d3 creatinine neat standard solutions were prepared to show equivalent LC-MS/MS response. The peak areas of 200 ng/mL and 40 ng/mL of both creatinine and d3 creatinine standard solutions were compared using the MRM transitions of 114/44 and 117/47, respectively. The results showed that the two solutions gave equivalent responses with mean percent difference and percent CV of less than 7.5%. See Table 1.

TABLE 1 D3 creatinine and creatinine equivalence using LC/MS/MS Std d3-Creatinine Creatinine CRN vs Percent (ng/ (MRM of (MRM of d3 CRN of D3 mL) 117/44) 114/44) % difference Response 40 791648 717010 10.4 90.6 40 804513 780182 3.1 97.0 40 774228 717528 7.9 92.7 40 776144 823064 −5.7 106.0 40 766927 828642 −7.4 108.0 40 741290 758937 −2.3 102.4 Mean 1.0 99.4 % CV 7.2 Std d3-Creatinine Creatinine CRN vs Percent (ng/ (MRM of (MRM of d3 CRN of D3 mL) 117/47) 114/44) % difference Response 200 3296195 3336107 −1.2 101.2 200 3469440 3325274 4.3 95.8 200 3416181 3428709 −0.4 100.4 200 3363696 3185389 5.6 94.7 200 3335259 3390463 −1.6 101.7 200 3255799 3321365 −2.0 102.0 Mean 0.8 99.3 % CV 3.2

These results show that d3 creatinine and creatinine give equivalent LC/MS/MS responses and d3-creatinine can be used as a surrogate analyte for creatinine. This is not surprising since deuterated compounds are used routinely as stable label internal standards, in regulated environments to validate assays. These deuterated standards have been shown to correct LC/MS/MS response of analyte from matrix effects as well as other extraction and chromatographic related effects. Since the only difference is an extra proton at three hydrogen atoms on the methyl group, we would expect the two compounds to behave almost identically throughout the extraction, chromatographic separation and mass spectral detection.

Determination of Isotope Ratio

This method is used to determine the amount of d3 creatinine in human urine that has been converted from a dose of d3 creatine. Additionally, the amount of endogenous creatinine will be determined using the d3 creatinine standard curve. The amount of endogenous creatinine is much greater (1000 fold) than the levels of d3-creatinine in the human clinical urine samples, therefore instead of diluting the sample, the M+2 isotope of creatinine will be monitored. This will allow the simultaneous measurement of creatinine and d3-creatinine from one sample using a urine matrix calibration curve. The peak area of the MRM of (M+2) endogenous creatinine (116/44) is monitored along with the d3 creatinine MRM of 117/47. A correction factor that represents the ratio of the MRM of 116/44 to 114/44, is used to correct the calculated concentrations determined from the d3-creatinine calibration curve.

The isotope ratio (response ratio) or difference in peak area response from the naturally abundant form of creatinine (M+0) or m/z=114 to the much less abundant form of creatinine (M+2) or m/z=116 is calculated experimentally. The isotope ratio is determined using two different experimental procedures. The original experimental design uses one standard concentration, a 200 ng/mL creatinine solution (Table 2a). The peak area of the creatinine is monitored at both the M+0 and M+2 MRM transitions (114/44 and 116/44), respectively. One solution was used to reduce variation which may occur from separate injections and preparation of separate solutions. This concentration is chosen because it allows the peak area of both MRMs to be in the detector range, and with adequate signal to noise for the smaller peak. However, some variability in the day to day measurements is observed (±10%) as shown in Table 3. Therefore, an additional experiment to generate this response ratio was performed.

In the second approach, the response ratio is experimentally determined using two separate solutions. A separate solution for each MRM transition is prepared which gives peak areas that are closer in magnitude to each other. A 10 ng/mL solution of creatinine is used to acquire the MRM transition of 114/44 and a 500 ng/mL solution is used to acquire the MRM transition of 116/44. These solutions are injected on the LC/MS/MS system in replicates of 10 and the mean peak area ratio (PAR) for each solution is determined. The response ratio is then calculated by dividing the mean PAR of 116/44 by the corrected PAR of 114/44. In order to compare the PARs from the two MRMs, the PAR from the 10 ng/mL solutions is multiplied by 50 (since 500 ng/mL is 50 times larger than the 10 ng/mL), an example is shown in Table 2b. This allows the peak area of both solutions to be closer in value and potentially eliminating errors associated with integrating peaks with vastly different signal to noise values.

Table 2 Creatinine Response Ratio (M+2/M+0) Determination Using LC/MS/MS

2a. Determined using a single creatinine standard solution Peak Area Ratio Creatinine Creatinine STD (M + 2) MRM (M + 0) MRM Response (ng/mL) of 116/44 of 114/44 Ratio 200 0.0209 9.19 0.00227 200 0.0216 9.42 0.00229 200 0.0195 9.53 0.00205 200 0.0208 9.42 0.00221 200 0.0202 9.37 0.00216 200 0.0199 9.46 0.00210 200 0.0188 9.22 0.00204 200 0.0201 9.64 0.00209 200 0.0202 9.33 0.00217 200 0.0188 9.49 0.00198 200 0.0200 9.45 0.00212 200 0.0198 9.32 0.00212 Mean 0.0201 9.4 0.00213 % CV 4.05 1.35 3.10

2b. Determined using separate creatinine concentrations Peak Area Ratio Creatinine Creatinine Creatinine (M + 2) MRM (M + 0) MRM (M + 0)* MRM Response of 116/44 of 114/44 of 114/44 Ratio 0.0460 0.4240 21.2 0.00217 0.0467 0.4240 21.2 0.00220 0.0466 0.3990 20.0 0.00234 0.0471 0.4110 20.6 0.00229 0.0477 0.4000 20.0 0.00239 0.0459 0.3850 19.3 0.00238 0.0453 0.3990 20.0 0.00227 0.0452 0.4000 20.0 0.00226 0.0443 0.3920 19.6 0.00226 0.0442 0.3780 18.9 0.00234 Mean 0.0459 0.4 20.1 0.00229 % CV 2.53 3.75 3.75 3.15 *= corrected for concentration difference

The corrected peak area ratio would be equivalent to a 500 ng/mL creatinine standard monitoring the peak area of the MRM transition of 114/44.

The isotope ratio (response ratio) was determined on multiple occasions over a four month time span and on two different triple quadrapole instruments. The mean of these nine values was determined and the inverse of this response ratio is the dilution factor used to correct the creatinine values in the LIMS system. See Table 3.

TABLE 3 Summary of Response Ratio (M + 2/M + 0) Determined using LC/MS/MS Date Response Ratio Instrument Name 22 Nov. 2011 0.00206 RTP12 29 Nov. 2011 0.00213 RTP12 AM 7 Dec. 2011 0.00193 RTP12 PM 7 Dec. 2011 0.00193 RTP12 * 14 Jan. 2012 0.00221 RTP12 * 16 Jan. 2012 0.00229 RTP12 * 17 Jan. 2012 0.00195 RTP12 * 7 Feb. 2012 0.00229 RTP12 * 10 Feb. 2012 0.00249 RTP52 Mean 0.002142 % CV 9.09 *= performed using two concentrations of creatinine

This experimentally determined response ratio is used to correct peak areas of creatinine M+2 (MRM of 116/44) and these corrected peak areas of creatinine were compared to peak areas run for the same concentration of d3 creatinine standard (MRM of 117/47). The comparison of the corrected creatinine peak area to the peak area obtained from the d3 creatinine standards gave equivalent responses with percent difference and percent CV of less than 10%. See Table 4.

TABLE 4 Creatinine (M + 2) Response Corrected using Response Ratio CRN CRN M + 2* d3 CRN STD (MRM 116/44) Corrected as to (MRM 117/47) Percent (ng/mL) M + 2 M + 0 Peak Area Difference 200 7856.2 3667693.7 3599411.5 98.1 200 7422.7 3465312.8 3682013.9 106.3 200 6101.3 2848412.7 3516609.5 123.5 200 7490.2 3496825.4 3330922.4 95.3 200 7288.0 3402427.6 3359823.2 98.7 200 6625.6 3093183.9 3264518.6 105.5 Mean 7130.7 3328976.0 3458883.2 104.6 % CV 9.0 9.0 4.8 9.8 *= corrected peak area (divided by the mean response ratio of 0.002142) 

1. A method of determining the concentration of creatinine in a biological sample from a subject, said method comprising the steps of: (a) obtaining a biological sample from the subject; (b) analyzing the biological sample to determine the peak area of the creatinine M+2 isotope peak for the biological sample; (c) comparing the peak area determined in step (b) to a calibration curve generated using D3-creatinine to determine the concentration of the creatinine M+2 isotope in the biological sample; (d) dividing the concentration obtained in step (c) by a dilution factor, where the dilution factor is the ratio of the concentration of creatinine M+2 to the concentration of creatinine M+0 in the biological sample.
 2. The method of claim 1, wherein said biological sample is a urine sample.
 3. The method of claim 1, wherein said biological sample is a blood sample.
 4. The method of claim 1, wherein said subject is human.
 5. The method of claim 1, where the peak area of the creatinine M+2 isotope peak is determined using liquid chromatography/mass spectroscopy (LC/MS/MS).
 6. The method of claim 1, wherein the dilution factor is 0.0002142±0.0000214.
 7. The method of claim 1 wherein the dilution factor is 0.0002142±0.00001.
 8. The method of claim 1 wherein the dilution factor is 0.0002142±0.000005.
 9. The method of claim 1 wherein D3-creatine has been administered to the subject and wherein the method further comprises the step of analyzing the biological sample to determine the peak area of D3-creatinine in the biological sample.
 10. A method of determining the total body skeletal muscle mass in a human subject, comprising the steps of: (a) orally administering 10-200 mg D3-creatine or a salt or hydrate thereof to the subject; (b) allowing at least 24 hours to elapse after the administration of the D3-creatine; (c) obtaining a biological sample from the subject, (d) determining the concentration of creatinine in said sample according to the method of claim 1; (e) determining the concentration of D3-creatinine in said biological sample; and (f) using the creatinine and D3-creatinine concentrations determined in steps (d) and (e) to calculate the total body skeletal muscle mass of the subject.
 11. The method of claim 9 wherein said subject is female and 20-40 mg of D3-creatine are administered.
 12. The method of claim 9 wherein said subject is male and 50-70 mg of D3-creatine are administered.
 13. The method of claim 9, wherein D3-creatine monohydrate is administered to the subject. 